This application claims the benefit of Korean Patent Application No. 2004-0090497, filed on Nov. 8, 2004, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention relates to a cell lysis method by an immobilized metal-ligand complex.
2. Description of the Related Art
Isolation methods of DNA from cells were provided using materials that have the proclivity of binding to DNA. Examples materials of isolation methods of DNA are silica, glass fiber, anion exchange resin and magnetic beads (Rudi, K. et al., Biotechniqures 22, 506-511 (1997); and Deggerdal, A. et al., Biotechniqures 22, 554-557 (1997)). To avoid the manual steps and to remove operator error, several automatic machines were developed for high-throughput DNA extraction.
Cell lysis is conventionally performed by a mechanical, chemical, thermal, electrical, ultrasonic, and microwave method (Michael T. Taylor et al., Anal.Chem., 73, 492-496 (2001)).
Heating is an alternative for disrupting cell walls or membranes. A disadvantage of simple heating is that it denatures proteins that can stick to the released DNA. They are likely to interfere with DNA amplification. A physical method is the use of a pressure apparatus, which is bulky and expensive, and thus, is not suitable for a Lab-On-a-Chip (LOC) application.
An ultrasonication is an alternative physical method; solutions or suspensions of cells are placed in a chamber placed in an ultrasonic bath. Ultrasonic disruption has many disadvantages in cell lysis. First of all, the energy distribution of ultrasonic is not uniform. The nonuniform distribution of ultrasonic energy leads to inconsistent results. Second, due to the energy divergence in the ultrasonic bath, it often takes several minutes to completely disrupt the cells. Lastly, ultrasonic method has unpleasant sound for human ears.
A chemical method includes the use of lysing agents for disrupting cells to release DNA. The lysing agents include detergents, alkaline treatments and chaotropic agents.
Detergents will disrupt the lipid bilayer allowing the contents to be released and will solubilize membrane proteins. It is most commonly used to lyse animal cells. Many detergents denature proteins. However, the addition of a cell lysis solution and the subsequent removal process are required and bubbles are generated. Thus, this method is not suitable for LOC implementation.
Alkaline treatments solubilize the phospholipid and protein components of the cell membrane. It is the easiest and least expensive chemical lysis method. Further, it is a fast, reliable and relatively clean way to obtain DNA from cells. However, the addition of an alkaline lysis solution for cell lysis and the neutralization process after lysis for DNA stabilization and the subsequent process application are required. Thus, steps upon LOC implementation are increased and the cell solution is diluted in direct proportion to the amount of the solution added.
Chaotropic agents having the ability to disrupt hydrophobic interactions may denature protein, but do not DNA or RNA. However, it is necessary to add a cell lysis solution to the chaotropic agent. Since the agent itself is very toxic and acts as a PCR inhibitor, the subsequent removal process is required. Thus, it is not suitable for LOC implementation.
Thus, the biggest problem in the conventional chemical lysis methods is that the addition of a lysis reagent allows a cell solution to be diluted, which results in an increase in the cell volume, and a separate removal process for removing the reagent should be performed after cell lysis is completed.
U.S. Pat. No. 5,304,487 discloses that physical protrusions in a microchannel or sharp-edged particles in a chamber or channel are used to mechanically lyse cells. Although there is a description of a solid support, there is no description of a method of lysing cells by immobilizing a metal-ligand complex on a solid support.
Thus, the inventors of the present invention discovered that a material having a cell lysis ability is immobilized on a solid surface to overcome the dilution problem resulting from the addition of the lysis solution and implement LOC without a separate process for removing the reagent in the course of studying a lysis method of cells or viruses on the basis of conventional technologies.